In the central nervous system (CNS) the transmission of stimuli takes place by the interaction of a neurotransmitter, which is sent out by a neuron, with a neuroreceptor. L-glutamic acid, the most commonly occurring neurotransmitter in the CNS, plays a critical role in a large number of physiological processes. The glutamate-dependent stimulus receptors are divided into two main groups. The first main group forms ligand-controlled ion channels, ionotropic glutamate receptors (iGLR). The metabotropic glutamate receptors (mGluR) form the second main group and, furthermore, belong to the family of G-protein-coupled receptors.
Glutamate receptors are involved in neuronal synapses in mammals and other animals, controlling functions such as learning and memory. Recent genomic sequencing and Expressed Sequence Tag analyses demonstrate that plants also possess orthologues of these receptors (Lam et al. 1998. Nature 396: 125-126). Furthermore an iGLR like receptor has also been reported in Cyanobacterium Synechosystis PCC 6803 (Chen et al. 1999. Nature 402:817) indicating that the putative GLRs are involved in well conserved sensing processes that has evolved into neuronal signaling in higher animals.
Based on ligand selectivity and ion conductance properties, mammalian iGLRs can be classified in to multiple classes,—amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)—kainite (KA), N-methyl-D-aspartate (NMDA) and Delta receptors (Sprengel and Seeburg, Handbook of receptors and channels: Ligand and voltage gated ion channels. CRC Press. Pp. 213-263, 1995).
At present, eight different members of mGluR are known and of these some even have sub-types. On the basis of structural parameters, the different influences on the synthesis of secondary metabolites and the different affinity to low-molecular weight chemical compounds, these eight receptors can be subdivided into three sub-groups: mGluR1 and mGluR5 belong to group I, mGluR2 and mGluR3 belong to group II and mGluR4, mGluR6, mGluR7 and mGluR8 belong to group III.
In Arabidopsis thaliana there are twenty of these receptor genes called glutamate receptors, that have been characterized by expression, and phylogenetic analyses (Chiu et al. 2002. Mol. Biol. Evol. 19:1066). Physiological analyses show that these receptors are involved in the regulation of C/N metabolism, calcium homeostasis, and stress responses (Kang and Turano. 2003. PNAS. 100:6872; Kim et al. 2001. Plant Cell Physiol. 42:74; Meyerhoff et al. 2005. Planta. 222:418).
Cell growth (accumulation of mass) is an extensively coordinated process that is regulated in both time and space. When nutrients and other appropriate growth stimuli are present, cells up regulate macromolecular synthesis and thereby increase in size and mass. Conversely, cells respond to nutrient limitation or other types of stress by restraining macromolecular synthesis and enhancing turnover of excess mass.
The target of rapamycin (TOR) is a conserved Ser/Thr kinase that regulates cell growth and metabolism in response to environmental cues. Every eukaryotic genome examined including yeasts, algae, slime mold, plants, worms, flies and mammals contain a TOR gene. Eukaryote TORs are large proteins (˜280 kDa) that share 40%-60% identity in their primary sequence and belong to a group of kinases known as the phosphatidylinositol kinase-related kinase (PIKK) family.
When growth conditions are favorable, TOR is active and yeast cells maintain a robust rate of ribosome biogenesis, translation initiation, and nutrient import. However, rapidly growing yeast cells treated with rapamycin, starved for nitrogen, or depleted of both TOR1 and TOR2 dramatically downregulate general protein synthesis, upregulate macroautophagy (the random sequestration and delivery of cytoplasm to the lysosome/vacuole), and activate several stress-responsive transcription factors. Growth factors, nutrients esp. amino acids, energy and stress regulate TOR pathway. However how amino acid levels are signaled to TOR is not fully understood. (Wullschleger et al. Cell. 2006. 124, 471).
Organisms modulate their growth according to nutrient availability. Although individual cells in multicellular organism including animal may respond directly to nutrient levels growth of entire organism needs to be co-coordinated. In Drosophila the coordination of organismal growth from the fat body is well studied. This involves TOR signaling in the fat body and a remote inhibition of organismal growth via local repression of PI3 kinase signaling in peripheral tissues (Colombani et al. Cell. 2003, 114, 739) In the rat, mammalian TOR (mTOR) in hypothalamus regulates food intake based on the level of amino acids (Cota et al. Science 2006. 312, 927).
N is an important macronutrient in modern agriculture with significant economic and ecological impact (Nanjing declaration on N management www.initrogen.org/nanjing_declaration.0.html). The yield of crop plants is directly related to the level of N applied. Modern crop production systems exploit plant breeding methods to develop high yielding, N responsive crop plants with increases in harvest index and biomass production (Hirel et al. 2001. PI. Phyiol. 125, 1258, Peng et al. 2000 Crop Sci. 40, 307). Genomic and physiological approaches in plant breeding are expected to provide further improvements in yield. For example, selection for improved photosynthetic rates and stomatal conductance are considered important physiological characteristics for yield improvement in wheat (Reynolds et al. 1999 Crop Sci. 39, 1611). Molecular approaches that include (i) over expression of key enzymes involved in N assimilation such as glutamine synthetase (GS) and asparagine synthetase (AS) in Arabidopsis and pea (U.S. Pat. No. 6,864,405), (ii) reduced expression of enzymes involved in cytokinin catabolism like an isoform of cytokinin oxidase in rice spiklets (Yanagisawa et al. Proc. Natl. Acad. Sci. USA. 2004. 101, 7833) and (iii) overexpression of transcription factor Dof1 in Arabidopsis (Ashikari et al. 2005. Science 309, 741) are shown to have improved biomass, N assimilation and yield. While N sensing is known to co-ordinate the up regulation of genes involved in many of the above mentioned pathways (Scheible et al. Plant Physiol. 2004. 136:2483), molecular components involved in the sensing processes remained elusive.
N is taken up by plants mostly in the form of nitrate or ammonium by specific nitrate or ammonium transporters, respectively. Upon uptake N is transformed into amino acids through the GS-GOGAT pathways (FIG. 1, G. M. Coruzzi. 2003. The Arabidopsis book). Plants transport N mostly as amino acids, though smaller amounts of nitrate and ammonia can be detected in the transporting vessels. There is tremendous interest in how plants sense the N status at the cellular and at whole plant level. The level of endogenous N regulates various processes including photosynthesis, major metabolic pathways, protein and DNA syntheses and growth and development (Scheible et al. Plant Physiol. 2004. 136:2483-99). Nitrate and ammonium are known to induce a set of genes that are involved in N uptake, assimilation pathways. However, inhibitors such as azaserine or methionine sulfoximine which inhibit the GS-GOGAT pathway, were shown to inhibit the nitrate induced signaling pathways (Vidamer et al. 2000 Plant Physiol. 123: 307, Kawachi et al. 2002. Physiol. Plantarum 114: 41). Plant orthologues of bacterial PII proteins, a regulator of bacterial N metabolism has been identified (U.S. Pat. Nos. 6,822,079, 6,177,275). However the role these proteins play in plant N sensing is not known. Similarly, Arabidopsis thaliana glutamate receptor 1.1 (AtGLR1.1) has been shown to be involved in the regulation of C and N metabolism (Kang and Turano. 2003. PNAS. 100:6872). Currently scientists do not have a clear understanding of the molecular components in N sensing in higher plants.
Currently with the available agricultural technologies the loss of applied N is estimated to be 60-70% (Raun, W R. and Johnson G V. 1999. Agronomy Journal. 91:357). Development of crop plants with improved N use efficiency has significant economic and ecological value. The yield level of crop plants is directly related to the level of N applied. Modern crop production systems exploit plant breeding methods to develop high yielding, N responsive crop plants with increased harvest index and biomass production (Hirel et al. 2001. PI. Phyiol. 125, 1258, Peng et al. 2000 Crop Sci. 40, 307). There is a need for technology to generate, and/or select plants with improved N use efficiency, biomass and yield, and plants with improved nutritional value. These traits are of economic importance. Presently plant biologists do not have a technology that can improve N sensing and yield, and thus there is a need for technology which provides a method to develop crop plants with higher N sensing capability that result in higher biomass and yield.